Polynucleotide primers and probes

A polynucleotide primer combination comprising a first polynucleotide and a second polynucleotide,

• the first polynucleotide (P) comprising (i) a first domain (Pa) at the 3′ end having a sequence that (a) does not comprise a blocking group, (b) is fully complementary to a sequence (T1) in a first target polynucleotide region, (c) is not fully complementary to a sequence (T1*) in a first non-target polynucleotide region, and (d) is about 5 to about 20 nucleotides in length, and (ii) a second domain (Pc) comprising a unique polynucleotide sequence, and
• the second polynucleotide (F) comprising (i) a first domain (Fb) having a sequence that is longer than Pa and that is complementary to a second target polynucleotide region (T2) and (ii) a second domain (Fd) comprising a polynucleotide sequence sufficiently complementary to Pc such that Pc and Fd will hybridize when Pa is hybridized to T1 and Fb is hybridized to T2, wherein F further comprises a blocking group attached at its 3′ end which blocks extension from a DNA polymerase,
• wherein P and/or F further comprise a modified nucleic acid, and
• wherein the sequence in the first region (T1) in the target polynucleotide differs at a base from the sequence in the first region (T1*) in the non-target polynucleotide.